Staphylococcus aureus is often the cause of nosocomial infections, particularly when these infections involve bacterial colonization of indwelling devices. The fact that these bacteria are frequently carried asymptomatically on the skin and mucosal surfaces of many individuals permits easy colonization of the pristine surfaces of such devices during implantation. Once biofilms grow on these surfaces, they are difficult to eradicate because the constituent cells become largely insensitive to the action of many commonly used antibiotics. These findings, combined with the fact that a large percentage of S. aureus isolates identified in infections are now resistant to numerous antibiotics makes it clear that new ways of combating staphylococcal biofilm-associated infections need to be developed. The goal of this project is to understand how methicillin-resistant S. aureus (MRSA) forms biofilms and thereby identify targets for the discovery of drugs that block or reverse the formation of surface-associated communities. To form a biofilm, S. aureus must produce an extracellular matrix. Major components of the matrix are proteins and DNA. Many of the proteins are cytoplasmic in origin and are thus moonlighting in their second role as components of the matrix. The extracellular DNA requires the matrix proteins in order to adhere to the cells, serving as an electrostatic net that holds the remaining cells together in the biofilm. Using a comprehensive molecular genetic approach, we identified genes needed for the release of extracellular DNA during biofilm formation, including the gene for a phosphodiesterase that controls the levels of a well-known second messenger, cyclic-di-AMP. We have shown that cyclic-di-AMP levels drop when cells enter the biofilm state. We propose to take advantage of a biosensor for measuring cyclic-di-AMP levels in cells to identify genes that control its levels as cells enter the biofilm state. We will take advantage of the discovery that mutants with altered levels of the second messenger are resistant to the dye Congo red to discover additional genes involved in controlling cyclic-di-AMP levels. We propose that the drop in cyclic-di-AMP levels weakens the cell envelope, causing some cells to lyse and liberate protein and DNA for incorporation into the matrix. We will investigate the hypothesis that low cyclic-di-AMP levels impair proper biosynthesis of teichoic acid, the cell envelope polymers needed for cell wall integrity. Finally, we exploit our fluorescent assay for c-di-AMP to carry out a cell-based screen for small molecules that cause levels of the second messenger to rise. These hits will be a starting point for the development of drugs that block biofilm formation.